Neutrokine-alpha

ABSTRACT

The present invention relates to a novel Neutrokine-α protein which is a member of the TNF protein family. In particular, isolated nucleic acid molecules are provided encoding the human Neutrokine-α protein including soluble forms of the extracellular domain. Neutrokine-α polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of Neutrokine-α activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.

[0001] This application is a divisional of U.S. application Ser. No.09/005,874 filed Jan. 12, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/036,100, filed Jan. 14,1997 and which is also a continuation-in-part of International PatentApplication No. PCT/US96/17957, filed Oct. 25, 1996, all of which areincorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel cytokine expressed byneutrophils which has therefore been designated Neutrokine-α protein(“Neutrokine-α”). In particular, isolated nucleic acid molecules areprovided encoding the Neutrokine-α protein. Neutrokine-α polypeptidesare also provided, as are vectors, host cells and recombinant methodsfor producing the same.

[0003] Related Art

[0004] Human tumor necrosis factors (TNF-α) and (TNF-β, or lymphotoxin)are related members of a broad class of polypeptide mediators, whichincludes the interferons, interleukins and growth factors, collectivelycalled cytokines (Beutler, B. and Cerami, A., Annu. Ret. Immunol.,7:625-655 (1989)). Sequence analysis of cytokine receptors has definedseveral subfamilies of membrane proteins (1) the Ig superfamily, (2) thehematopoietin (cytokine receptor superfamily and (3) the tumor necrosisfactor (TNF)/nerve growth factor (NGF) receptor superfamily (for reviewof TNF superfamily see, Gruss and Dower, Blood 85(12):3378-3404 (1995)and Aggarwal and Natarajan, Eur. Cytokine Netw., 7(2):93-124 (1996)).The TNF/NGF receptor superfamily contains at least 10 differenceproteins. Gruss and Dower, supra. Ligands for these receptors have beenidentified and belong to at least two cytokine superfamilies. Gruss andDower, supra.

[0005] Tumor necrosis factor (a mixture of TNF-α and TNF-β) wasoriginally discovered as a result of its anti-tumor activity, however,now it is recognized as a pleiotropic cytokine capable of numerousbiological activities including apoptosis of some transformed celllines, mediation of cell activation and proliferation and also asplaying important roles in immune regulation and inflammation.

[0006] To date, known members of the TNF-ligand superfamily includeTNF-α, TNF-β (lymphotoxin-α), LT-β, OX40L, Fas ligand, CD30L, CD27L,CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic,TNF-like molecules with approximately 20% sequence homology in theextracellular domains (range, 12%-36%) and exist mainly asmembrane-bound forms with the biologically active form being atrimeric/multimeric complex. Soluble forms of the TNF ligand superfamilyhave only been identified so far for TNF, LT-β, and Fas ligand (for ageneral review, see Gruss, H. and Dower, S. K., Blood, 85(12):3378-3404(1995)), which is hereby incorporated by reference in its entirety.These proteins are involved in regulation of cell proliferation,activation, and differentiation, including control of cell survival ordeath by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin.Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).

[0007] Tumor necrosis factor-alpha (TNF-α; also termed cachectin;hereinafter “TNF”) is secreted primarily by monocytes and macrophages inresponse to endotoxin or other stimuli as a soluble homotrimer of 17 kDprotein subunits (Smith, R. A. et al., J. Biol. Chem. 262:6951-6954(1987)). A membrane-bound 26 kD precursor form of TNF has also beendescribed (Kriegler, M. et al., Cell 53:45-53 (1988)).

[0008] Accumulating evidence indicates that TNF is a regulatory cytokinewith pleiotropic biological activities. These activities include:inhibition of lipoprotein lipase synthesis (“cachectin” activity)(Beutler, B. et al., Nature 316:552 (1985)), activation ofpolymorphonuclear leukocytes (Klebanoff, S. J. et al., J. Immunol.136:4220 (1986); Perussia, B., et al., J. Immunol. 138:765 (1987)),inhibition of cell growth or stimulation of cell growth (Vilcek, J. etal., J. Exp. Med. 163:632 (1986); Sugarman, B. J. et al., Science230:943 (1985); Lachman, L. B. et al., J. Immunol. 138:2913 (1987)),cytotoxic action on certain transformed cell types (Lachman, L. B. etal., supra; Darzynkiewicz, Z. et al., Canc. Res. 44:83 (1984)),antiviral activity (Kohase, M. et al., Cell 45:659 (1986); Wong, G. H.W. et al., Nature 323:819 (1986)), stimulation of bone resorption(Bertolini, D. R. et al., Nature 319:516 (1986); Saklatvala, J., Nature322:547 (1986)), stimulation of collagenase and prostaglandin E2production (Dayer, J.-M. et al., J. Exp. Med. 162:2163 (1985)); andimmunoregulatory actions, including activation of T cells (Yokota, S. etal., J. Immunol. 140:531 (1988)), B cells (Kehrl, J. H. et al., J. Exp.Med. 166:786 (1987)), monocytes (Philip, R. et al., Nature 323:86(1986)), thymocytes (Ranges, G. E. et al., J. Exp. Med. 167:1472(1988)), and stimulation of the cell-surface expression of majorhistocompatibility complex (MHC) class I and class II molecules(Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446 (1986);Pujol-Borrel, R. et al., Nature 326:304 (1987)).

[0009] TNF is noted for its pro-inflammatory actions which result intissue injury, such as induction of procoagulant activity on vascularendothelial cells (Pober, J. S. et al., J. Immunol. 136:1680 (1986)),increased adherence of neutrophils and lymphocytes (Pober, J. S. et al.,J. Immunol. 138:3319 (1987)), and stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).

[0010] Recent evidence implicates TNF in the pathogenesis of manyinfections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immunedisorders, neoplastic pathology, e.g., in cachexia accompanying somemalignancies (Oliff, A. et al., Cell 50:555 (1987)), and in autoimmunepathologies and graft-versus host pathology (Piguet, P.-F. et al., J.Exp. Med. 166:1280 (1987)). The association of TNF with cancer andinfectious pathologies is often related to the host's catabolic state. Amajor problem in cancer patients is weight loss, usually associated withanorexia. The extensive wasting which results is known as “cachexia”(Kern, K. A. et al. J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexiaincludes progressive weight loss, anorexia, and persistent erosion ofbody mass in response to a malignant growth. The cachectic state is thusassociated with significant morbidity and is responsible for themajority of cancer mortality. A number of studies have suggested thatTNF is an important mediator of the cachexia in cancer, infectiouspathology, and in other catabolic states.

[0011] TNF is thought to play a central role in the pathophysiologicalconsequences of Gram-negative sepsis and endotoxic shock (Michie, H. R.et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., SecondVienna Shock Forum, p.463-466 (1989); Simpson, S. Q. et al., Crit. CareClin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.Endotoxin is a potent monocyte/macrophage activator which stimulatesproduction and secretion of TNF (Kornbluth, S. K. et al., J. Immunol.137:2585-2591 (1986)) and other cytokines. Because TNF could mimic manybiological effects of endotoxin, it was concluded to be a centralmediator responsible for the clinical manifestations ofendotoxin-related illness. TNF and other monocyte-derived cytokinesmediate the metabolic and neurohormonal responses to endotoxin (Michie,H. R. et al., N. Eng. J. Med. 318:1481-1486 (1988)). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, A. et al., Arch. Surg. 123:162-170(1988)). Elevated levels of circulating TNF have also been found inpatients suffering from Gram-negative sepsis (Waage, A. et al., Lancet1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p.715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17:489-497(1989); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990)).

[0012] Passive immunotherapy directed at neutralizing TNF may have abeneficial effect in Gram-negative sepsis and endotoxemia, based on theincreased TNF production and elevated TNF levels in these pathologystates, as discussed above. Antibodies to a “modulator” material whichwas characterized as cachectin (later found to be identical to TNF) weredisclosed by Cerami et al. (EPO Patent Publication 0,212,489, Mar. 4,1987). Such antibodies were said to be useful in diagnostic immunoassaysand in therapy of shock in bacterial infections. Rubin et al. (EPOPatent Publication 0,218,868, Apr. 22, 1987) disclosed monoclonalantibodies to human TNF, the hybridomas secreting such antibodies,methods of producing such antibodies, and the use of such antibodies inimmunoassay of TNF. Yone et al. (EPO Patent Publication 0,288,088, Oct.26, 1988) disclosed anti-TNF antibodies, including mAbs, and theirutility in immunoassay diagnosis of pathologies, in particularKawasaki's pathology and bacterial infection. The body fluids ofpatients with Kawasaki's pathology (infantile acute febrilemucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:178 (1967);Kawasaki, T., Shonica (Pediatrics) 26:935 (1985)) were said to containelevated TNF levels which were related to progress of the pathology(Yone et al., supra).

[0013] Other investigators have described mAbs specific for recombinanthuman TNF which had neutralizing activity in vitro (Liang, C-M. et al.Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, A. et al.,Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987);Bringman, T S et al., Hybridoma 6:489-507 (1987); Hirai, M. et al., J.Immunol. Meth. 96:57-62 (1987); Moller, A. et al. (Cytokine 2:162-169(1990)). Some of these mAbs were used to map epitopes of human TNF anddevelop enzyme immunoassays (Fendly et al., supra; Hirai et al., supra;Moller et al., supra) and to assist in the purification of recombinantTNF (Bringman et al., supra). However, these studies do not provide abasis for producing TNF neutralizing antibodies that can be used for invivo diagnostic or therapeutic uses in humans, due to immunogenicity,lack of specificity and/or pharmaceutical suitability.

[0014] Neutralizing antisera or mAbs to TNF have been shown in mammalsother than man to abrogate adverse physiological changes and preventdeath after lethal challenge in experimental endotoxemia and bacteremia.This effect has been demonstrated, e.g., in rodent lethality assays andin primate pathology model systems (Mathison, J. C. et al., J. Clin.Invest. 81:1925-1937 (1988); Beutler, B. et al., Science 229:869-871(1985); Tracey, K. J. et al., Nature 330:662-664 (1987); Shimamoto, Y.et al., Immunol. Lett. 17:311-318 (1988); Silva, A. T. et al., J.Infect. Dis. 162:421-427 (1990); Opal, S. M. et al., J. Infect. Dis.161:1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279-292(1990)).

[0015] To date, experience with anti-TNF mAb therapy in humans has beenlimited but shows beneficial therapeutic results, e.g., in arthritis andsepsis. See, e.g., Elliott, M. J. et al., Baillieres Clin. Rheumatol.9:633-52 (1995); Feldmann M, et al., Ann. N.Y. Acad. Sci. USA 766:272-8(1995); van der Poll, T. et al., Shock 3:1-12 (1995); Wherry et al.,Crit. Care. Med. 21:S436-40 (1993); Tracey K. J., et al., Crit. CareMed. 21:S415-22 (1993).

[0016] Mammalian development is dependent on both the proliferation anddifferentiation of cells as well as programmed cell death which occursthrough apoptosis (Walker, et al., Methods Achiev. Exp. Pathol. 13:18(1988). Apoptosis plays a critical role in the destruction of immunethymocytes that recognize self antigens. Failure of this normalelimination process may play a role in autoimmune diseases (Gammon etal., Immunology Today 12:193 (1991)).

[0017] Itoh et al. (Cell 66:233 (1991)) described a cell surfaceantigen, Fas/CD23 that mediates apoptosis and is involved in clonaldeletion of T-cells. Fas is expressed in activated T-cells, B-cells,neutrophils and in thymus, liver, heart and lung and ovary in adult mice(Watanabe-Fukunaga et al., J. Immunol. 148:1274 (1992)) in addition toactivated T-cells, B-cells, neutorophils. In experiments where amonoclonal Ab is cross-linked to Fas, apoptosis is induced (Yonehara etal., J. Exp. Med. 169:1747 (1989); Trauth et al., Science 245:301(1989)). In addition, there is an example where binding of a monoclonalAb to Fas is stimulatory to T-cells under certain conditions (Aldersonet al., J. Exp. Med. 178:2231 (1993)).

[0018] Fas antigen is a cell surface protein of relative MW of 45 Kd.Both human and murine genes for Fas have been cloned byWatanabe-Fukunaga et al., (J. Immunol. 148:1274 (1992)) and Itoh et al.(Cell 66:233 (1991)). The proteins encoded by these genes are bothtransmembrane proteins with structural homology to the Nerve GrowthFactor/Tumor Necrosis Factor receptor superfamily, which includes twoTNF receptors, the low affinity Nerve Growth Factor receptor and CD40,CD27, CD30, and OX40.

[0019] Recently the Fas ligand has been described (Suda et al., Cell75:1169 (1993)). The amino acid sequence indicates that Fas ligand is atype II transmembrane protein belonging to the TNF family. Thus, the Fasligand polypeptide comprises three main domains: a short intracellulardomain at the amino terminal end and a longer extracellular domain atthe carboxy terminal end, connected by a hydrophobic transmembranedomain. Fas ligand is expressed in splenocytes and thymocytes,consistent with T-cell mediated cytotoxicity. The purified Fas ligandhas a MW of 40 kD.

[0020] Recently, it has been demonstrated that Fas/Fas ligandinteractions are required for apoptosis following the activation ofT-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature373:441 (1995)). Activation of T-cells induces both proteins on the cellsurface. Subsequent interaction between the ligand and receptor resultsin apoptosis of the cells. This supports the possible regulatory rolefor apoptosis induced by Fas/Fas ligand interaction during normal immuneresponses.

[0021] Accordingly, there is a need to provide cytokines similar to TNFthat are involved in pathological conditions. Such novel cytokines couldbe used to make novel antibodies or other antagonists that bind theseTNF-like cytokines for therapy of disorders related to TNF-likecytokines.

SUMMARY OF THE INVENTION

[0022] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a cytokine that is structurallysimilar to TNF and related cytokines and is believed to have similarbiological effects and activities. This cytokine is named Neutrokine-αand the invention includes Neutrokine-α polypeptides having at least aportion of the amino acid sequence in FIGS. 1A and B (SEQ ID NO:2) oramino acid sequence encoded by the cDNA clone deposited in a bacterialhost on Oct. 22, 1996 assigned ATCC number 97768. The nucleotidesequence determined by sequencing the deposited Neutrokine-α clone,which is shown in FIGS. 1A and B (SEQ ID NO:1), contains an open readingframe encoding a complete polypeptide of 285 amino acid residuesincluding an N-terminal methionine, a predicted intracellular domain ofabout 46 amino acid residues, a predicted transmembrane domain of about26 amino acids, a predicted extracellular domain of about 213 aminoacids, and a deduced molecular weight for the complete protein of about31 kDa. As for other type II transmembrane proteins, soluble forms ofNeutrokine-α include all or a portion of the extracellular domaincleaved from the transmembrane domain and a polypeptide comprising thecomplete Neutrokine-α polypeptide lacking the transmembrane domain,i.e., the extracellular domain linked to the intracellular domain.

[0023] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a full-length Neutrokine-α polypeptide having the completeamino acid sequence in FIGS. 1A and B (SEQ ID NO:2) or as encoded by thecDNA clone contained in the ATCC Deposit of Oct. 22, 1996 ATCC Number97768; (b) a nucleotide sequence encoding the predicted extracellulardomain of the Neutrokine-α polypeptide having the amino acid sequence atpositions 73 to 285 in FIGS. 1A and B (SEQ ID NO:2) or as encoded by thecDNA clone contained in ATCC No. 97768 deposited on Oct. 22, 1996; (c) anucleotide sequence encoding a polypeptide comprising the Neutrokine-αintracellular domain (amino acid residues from about 1 to about 46 inFIGS. 1A and B (SEQ ID NO:2)) or as encoded by the cDNA clone containedin ATCC No. 97768 deposited on Oct. 22, 1996; (d) a nucleotide sequenceencoding a polypeptide comprising the Neutrokine-α transmembrane domain(amino acid residues from about 47 to about 72 in FIGS. 1A and B (SEQ IDNO:2) or as encoded by the cDNA clone contained in ATCC No. 97768deposited on Oct. 22, 1996; (e) a nucleotide sequence encoding a solubleNeutrokine a polypeptide having the extracellular and intracellulardomains but lacking the transmembrane domain; and (f) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d) or (e) above.

[0024] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 96%,97%, 98% or 99% identical, to any of the nucleotide sequences in (a),(b), (c), (d), (e) or (f) above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide in (a),(b), (c), (d), (e) or (f) above. This polynucleotide which hybridizesdoes not hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a Neutrokine-α polypeptide having an aminoacid sequence in (a), (b), (c), (d) or (e) above. A further nucleic acidembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of aNeutrokine-α polypeptide having an amino acid sequence which contains atleast one amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a polynucleotide which encodes the aminoacid sequence of a Neutrokine-α polypeptide to have an amino acidsequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1amino acid substitutions. Conservative substitutions are preferable.

[0025] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of Neutrokine-α polypeptides or peptides by recombinanttechniques.

[0026] The invention further provides an isolated Neutrokine-αpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the full-lengthNeutrokine-α polypeptide having the complete amino acid sequence shownin FIGS. 1A and B (SEQ ID NO:2) or as encoded by the cDNA clonecontained in ATCC No. 97768 deposited on Oct. 22, 1996; (b) the aminoacid sequence of the predicted extracellular domain of the Neutrokine-αpolypeptide having the amino acid sequence at positions 73 to 285 inFIGS. 1A and B (SEQ ID NO:2) or as encoded by the cDNA clone containedin ATCC No. 97768 deposited on Oct. 22, 1996; (c) the amino acidsequence of the Neutrokine-α intracellular domain (amino acid residuesfrom about 1 to about 46 in FIGS. 1A and B (SEQ ID NO:2)) or as encodedby the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,1996; (d) the amino acid sequence of the Neutrokine-α transmembranedomain (amino acid residues from about 47 to about 72 in FIGS. 1A and B(SEQ ID NO:2)) or as encoded by the cDNA clone contained in ATCC No.97768 deposited on Oct. 22, 1996; and (e) the amino acid sequence of thesoluble Neutrokine-α polypeptide having the extracellular andintracellular domains but lacking the transmembrane domain, wherein eachof these domains is defined above.

[0027] The polypeptides of the present invention also includepolypeptides having an amino acid sequence with at least 90% similarity,and more preferably at least 95% similarity to those described in (a),(b), (c), (d) or (e) above, as well as polypeptides having an amino acidsequence at least 80% identical, more preferably at least 90% identical,and still more preferably 95%, 96%, 97%, 98% or 99% identical to thoseabove.

[0028] An additional embodiment of this aspect of the invention relatesto a peptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a Neutrokine-α polypeptide having an aminoacid sequence described in (a), (b), (c), (d) or (e) above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a Neutrokine-α polypeptide of the invention include portionsof such polypeptides with at least six or seven, preferably at leastnine, and more preferably at least about 30 amino acids to about 50amino acids, although epitope-bearing polypeptides of any length up toand including the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention. In anotherembodiment, the invention provides an isolated antibody that bindsspecifically to an polypeptide having an amino acid sequence describedin (a), (b), (c), (d) or (e) above.

[0029] The invention further provides methods for isolating antibodiesthat bind specifically to a Neutrokine-α polypeptide having an aminoacid sequence as described herein. Such antibodies are usefuldiagnostically or therapeutically as described below.

[0030] The invention also provides for pharmaceutical compositionscomprising soluble Neutrokine-α polypeptides, particularly humanNeutrokine-α polypeptides, which may be employed, for instance, to treattumor and tumor metastasis, infections by bacteria, viruses and otherparasites, immunodeficiencies, inflammatory diseases, lymphadenopathy,autoimmune diseases, graft versus host disease and to stimulateperipheral tolerance, destroy some transformed cell lines, mediate cellactivation and proliferation, and are functionally linked as primarymediators of immune regulation and inflammatory responses.

[0031] The invention further provides compositions comprising aNeutrokine-α polynucleotide or a Neutrokine-α polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprise aNeutrokine-α polynucleotide for expression of a Neutrokine-α polypeptidein a host organism for treatment of disease. Particularly preferred inthis regard is expression in a human patient for treatment of adysfunction associated with aberrant endogenous activity of aNeutrokine-α gene.

[0032] The present invention also provides a screening method foridentifying compounds capable of enhancing or inhibiting a cellularresponse induced by Neutrokine-α which involves contacting cells whichexpress Neutrokine-α with the candidate compound, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made in absence ofthe candidate compound; whereby, an increased cellular response over thestandard indicates that the compound is an agonist and a decreasedcellular response over the standard indicates that the compound is anantagonist.

[0033] In another aspect, a method for identifying Neutrokine-αreceptors is provided, as well as a screening assay for agonists andantagonists using such receptors. This assay involves determining theeffect a candidate compound has on Neutrokine-α binding to theNeutrokine-α receptor. In particular, the method involves contacting aNeutrokine-α receptor with a Neutrokine-α polypeptide and a candidatecompound and determining whether Neutrokine-α polypeptide binding to theNeutrokine-α receptor is increased or decreased due to the presence ofthe candidate compound. The antagonists may be employed to preventseptic shock, inflammation, cerebral malaria, activation of the HIVvirus, graft-host rejection, bone resorption, rheumatoid arthritis andcachexia (wasting or malnutrition)

[0034] The present inventors have discovered that Neutrokine-α isexpressed not only in neutrophils, but also in kidney, lung, peripheralleukocyte, bone marrow, T cell lymphoma, B cell lymphoma, activated Tcells, stomach cancer, smooth muscle, macrophages, and cord bloodtissue. For a number of disorders of these tissues and cells, such astumor and tumor metastasis, infection of bacteria, viruses and otherparasites, immunodeficiencies, septic shock, inflammation, cerebralmalaria, activation of the HIV virus, graft-host rejection, boneresorption, rheumatoid arthritis and cachexia (wasting or malnutrition,it is believed that significantly higher or lower levels of Neutrokine-αgene expression can be detected in certain tissues (e.g., bone marrow)or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to a“standard” Neutrokine-α gene expression level, i.e., the Neutrokine-αexpression level in tissue or bodily fluids from an individual nothaving the disorder. Thus, the invention provides a diagnostic methoduseful during diagnosis of a disorder, which involves: (a) assayingNeutrokine-α gene expression level in cells or body fluid of anindividual; (b) comparing the Neutrokine-α gene expression level with astandard Neutrokine-α gene expression level, whereby an increase ordecrease in the assayed Neutrokine-α gene expression level compared tothe standard expression level is indicative of a disorder.

[0035] An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of Neutrokine-αactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedNeutrokine-α polypeptide of the invention or an agonist thereof.

[0036] A still further aspect of the invention is related to a methodfor treating an individual in need of a decreased level of Neutrokine-αactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of anNeutrokine-α antagonist. Preferred antagonists for use in the presentinvention are Neutrokine-α-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIGS. 1A and B show the nucleotide (SEQ ID NO:1) and deduced aminoacid (SEQ ID NO:2) sequences of the Neutrokine-α protein. Amino acids 1to 46 represent the intracellular domain, amino acids 47 to 72 thetransmembrane domain (the underlined sequence), and amino acids 73 to285, the extracellular domain (the remaining sequence).

[0038] FIGS. 2A-C show the regions of identity between the amino acidsequences of the Neutrokine-α protein (SEQ ID NO:2) and TNF-α (SEQ IDNO:3), TNF-β (lymphotoxin; SEQ ID NO:4) and FAS ligand (SEQ ID NO:5),determined by the “Megalign” routine which is part of the computerprogram called “DNAStar”, Amino acid residues that match the consensussequence are shaded.

[0039]FIG. 3 shows an analysis of the Neutrokine-α amino acid sequence(SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown. In the “Antigenic Index—Jameson-Wolf”graph, the indicate location of the highly antigenic regions of theNeutrokine-α protein, i.e., regions from which epitope-bearing peptidesof the invention may be obtained.

[0040] FIGS. 4A-C show the alignment of the Neutrokine-α nucleotidesequence (SEQ ID NO:1) determined from the human cDNA deposited in ATCCNo. 97768 deposited on Oct. 22, 1996 with related human cDNA clones ofthe invention which have been designated HSOAD55 (SEQ ID NO:7), HSLAH84(SEQ ID NO:8) and HLTBM08 (SEQ ID NO:9).

DETAILED DESCRIPTION

[0041] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding Neutrokine-α polypeptide having theamino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2), which wasdetermined by sequencing a cloned cDNA Neutrokine-α. The nucleotidesequence shown in FIGS. 1A and B (SEQ ID NO:1) was obtained bysequencing the HNEDU15 clone, which was deposited on Oct. 22, 1996 atthe American Type Culture Collection, 10801 University Drive, Manassas,Va. 20110-2209 and assigned ATCC Deposit No. 97768. The deposited cloneis contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla,Calif.).

[0042] The Neutrokine-α protein of the present invention shares sequencehomology with the translation product of the human mRNAs for TNF-α,TNF-β and Fas ligand (FIGS. 2A-C). As noted above, TNF-α is thought tobe an important cytokine that plays a role in cytotoxicity, necrosis,apoptosis, costimulation, proliferation, lymph node formation,immunoglobulin class switch, differentiation, antiviral activity,regulation of adhesion molecules and other cytokines and growth factors.

Nucleic Acid Molecules

[0043] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.,Foster City, Calif.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were predicted by translationof a DNA sequence determined as above. Therefore, as is known in the artfor any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart. As is also known in the art, a single insertion or deletion in adetermined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded by the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

[0044] By “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U).

[0045] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A and B, a nucleic acid molecule of the presentinvention encoding a Neutrokine-α polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A and B (SEQ ID NO:1) wasdiscovered in a cDNA library derived from neutrophils. Expressedsequence tags corresponding to a portion of the Neutrokine-α cDNA werealso found in kidney, lung, peripheral leukocyte, bone marrow, T celllymphoma, B cell lymphoma, activated T cells, stomach cancer, smoothmuscle, macrophages, and cord blood tissue.

[0046] The Neutrokine-α gene contains an open reading frame encoding aprotein of about 285 amino acid residues, an intracellular domain ofabout 46 amino acids (amino acid residues from about 1 to about 46 inFIG. 1 (SEQ ID NO:2)), a transmembrane domain of about 26 amino acids(underlined amino acid residues from about 47 to about 72 in FIGS. 1Aand B (SEQ ID NO:2)), an extracellular domain of about 213 amino acids(amino acid residues from about 73 to about 285 in FIGS. 1A and B (SEQID NO:2)); and a deduced molecular weight of about 31 kDa. TheNeutrokine-α protein shown in FIGS. 1A and B (SEQ ID NO: 2) is about 20%similar and about 10% identical to human TNF-α which can be accessed onGenBank as Accession No. 339764.

[0047] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, the actual completeNeutrokine-α polypeptide encoded by the deposited cDNA, which comprisesabout 285 amino acids, may be somewhat shorter. In particular, thedetermined Neutrokine-α coding sequence contains a second methioninecodon which may serve as an alternative start codon for translation ofthe open reading frame, at nucleotide positions 210-213 in FIGS. 1A andB (SEQ ID NO:1). More generally, the actual open reading frame may beanywhere in the range of ±20 amino acids, more likely in the range of±10 amino acids, of that predicted from either the first or secondmethionine codon from the N-terminus shown in FIGS. 1A and B (SEQ IDNO:1). It will further be appreciated that, depending on the analyticalcriteria used for identifying various functional domains, the exact“address” of the extracelluar, intracelluar and transmembrane domains ofthe Neutrokine-α polypeptide may differ slightly. For example, the exactlocation of the Neutrokine-α extracellular domain in FIGS. 1A and B (SEQID NO:2) may vary slightly (e.g., the address may “shift” by about 1 toabout 20 residues, more likely about 1 to about 5 residues) depending onthe criteria used to define the domain. In this case, the ends of thetransmembrane domain and the beginning of the extracellular domain werepredicted on the basis of the identification of the hydrophobic aminoacid sequence in the above indicated positions, as shown in FIG. 3. Inany event, as discussed further below, the invention further providespolypeptides having various residues deleted from the N-terminus of thecomplete polypeptide, including polypeptides lacking one or more aminoacids from the N-terminus of the extracellular domain described herein,which constitute soluble forms of the extracellular domain of theNeutrokine-α protein.

[0048] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0049] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0050] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) with an initiationcodon at positions 147-149 of the nucleotide sequence shown in FIGS. 1Aand B (SEQ ID NO:1). In addition, isolated nucleic acid molecules of theinvention include DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the Neutrokine-α protein. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate the degenerate variants described above.In another aspect, the invention provides isolated nucleic acidmolecules encoding the Neutrokine-α polypeptide having an amino acidsequence encoded by the cDNA contained in the plasmid deposited on Oct.22, 1996. Preferably, this nucleic acid molecule will comprise asequence encoding the extracellular domain of the polypeptide encoded bythe above-described deposited cDNA clone.

[0051] The invention further provides an isolated nucleic acid moleculehaving the nucleotide sequence shown in FIGS. 1A and B (SEQ ID NO:1) orthe nucleotide sequence of the Neutrokine-α cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the Neutrokine-α gene in human tissue, for instance, byNorthern blot analysis.

[0052] The invention also provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HSOAD55(SEQ ID NO:7), HSLAH84 (SEQ ID NO:8), and HLTBM08 (SEQ ID NO:9).

[0053] The present invention is further directed to nucleic acidmolecules encoding portions of the nucleotide sequences described hereinas well as to fragments of the isolated nucleic acid molecules describedherein. In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 147-1001 of SEQ ID NO:1.

[0054] Further, the invention includes a polynucleotide comprising asequence at least 95% identical to any portion of at least about 30contiguous nucleotides, preferably at least about 50 nucleotides, of thesequence from nucleotide 1 to nucleotide 1082 in FIGS. 1A and B (SEQ IDNO:1), preferably excluding the nucleotide sequences determined from theabovelisted cDNA clones and the nucleotide sequences from nucleotide 797to 1082, 810 to 1082, and 346 to 542. More preferably, the inventionincludes a polynucleotide comprising nucleotide residues 147-500,147-450, 147-400, 147, 350, 200-500, 200-450, 200-400, 200-350, 250-500,250-450, 250-400, 250-350, 300-500, 300-450, 300-400, 300-350, 350-750,350-700, 350-650, 350-600, 350-550, 400-750, 400-700, 400-650, 400-600,400-550, 425-750, 425-700, 425-650, 425-600, 425-550, 450-1020,450-1001, 450-950, 450-900, 450-850, 450-800, 450-775, 500-1001,500-950, 500-900, 500-850, 500-800, 500-775, 550-1001, 550-950, 550-900,550-850, 550-800, 550-775, 600-1001, 600-950, 600-900, 600-850, 600-800,600-775, 650-1001, 650-950, 650-900, 650-850, 650-800, 650-775,700-1001, 700-950, 700-900, 700-850, 700-800, 700-775, 825-1082,850-1082, 875-1082, 900-1082, 925-1082, 950-1082, 975-1082, 1000-1082,1025-1082, and 1050-1082.

[0055] More generally, by a fragment of an isolated nucleic acidmolecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in FIGS. 1A and B (SEQ ID NO:1) is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-300 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in FIGS. 1A and B(SEQ ID NO:1). By a fragment at least 20 nt in length, for example, isintended fragments which include 20 or more contiguous bases from thenucleotide sequence of the deposited cDNA or the nucleotide sequence asshown in FIGS. 1A and B (SEQ ID NO:1). Preferred nucleic acid fragmentsof the present invention include nucleic acid molecules encodingepitope-bearing portions of the Neutrokine-α polypeptide as identifiedin FIGS. 1A and B and described in more detail below.

[0056] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22, 1996.By “stringent hybridization conditions” is intended overnight incubationat 42° C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl,75 mM trisodium cirate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0057] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 (e.g., 50) nt of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below.

[0058] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A and B (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the Neutrokine-α cDNAshown in FIGS. 1A and B (SEQ ID NO:1)), or to a complementary stretch ofT (or U) residues, would not be included in a polynucleotide of theinvention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly(A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

[0059] As indicated, nucleic acid molecules of the present inventionwhich encode a Neutrokine-α polypeptide may include, but are not limitedto those encoding the amino acid sequence of the extracellular domain ofthe polypeptide, by itself; and the coding sequence for theextracellular domain of the polypeptide and additional sequences, suchas those encoding the intracellular and transmembrane domain sequences,or a pre-, or pro- or prepro-protein sequence; the coding sequence ofthe extracellular domain of the polypeptide, with or without theaforementioned additional coding sequences.

[0060] Also encoded by nucleic acids of the invention are the aboveprotein sequences together with additional, non-coding sequences,including for example, but not limited to introns and non-coding 5′ and3′ sequences, such as the transcribed, non-translated sequences thatplay a role in transcription, mRNA processing, including splicing andpolyadenylation signals, for example, ribosome binding and stability ofmRNA; an additional coding sequence which codes for additional aminoacids, such as those which provide additional functionalities.

[0061] Thus, the sequence encoding the polypeptide may be fused to amarker sequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the Neutrokine-αfused to Fc at the N- or C-terminus.

[0062] Variant and Mutant Polynucleotides

[0063] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the Neutrokine-α protein. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

[0064] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the Neutrokine-α protein or portionsthereof. Also especially preferred in this regard are conservativesubstitutions.

[0065] Most highly preferred are nucleic acid molecules encoding theextracellular domain of the protein having the amino acid sequence shownin FIGS. 1A and B (SEQ ID NO:2) or the extracellular domain of theNeutrokine-α amino acid sequence encoded by the deposited cDNA clone.Further embodiments include an isolated nucleic acid molecule comprisinga polynucleotide having a nucleotide sequence at least 90% identical,and more preferably at least 95%, 96%, 97%, 98% or 99% identical to apolynucleotide selected from the group consisting of: (a) a nucleotidesequence encoding the Neutrokine-α polypeptide having the complete aminoacid sequence in FIGS. 1A and B (SEQ ID NO:2); (b) a nucleotide sequenceencoding the predicted extracellular domain of the Neutrokine-αpolypeptide having the amino acid sequence at positions 73-285 in FIGS.1A and B (SEQ ID NO:2); (c) a nucleotide sequence encoding theNeutrokine-α polypeptide having the complete amino acid sequence encodedby the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,1996; (d) a nucleotide sequence encoding the extracellular domain of theNeutrokine-α polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC No. 97768 deposited on Oct. 22, 1996; and(e) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c) or (d) above.

[0066] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aNeutrokine-α polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theNeutrokine-α polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0067] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIGS. 1A and B or to thenucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0068] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIGS. 1A and B (SEQ ID NO:1) or to the nucleic acidsequence of the deposited cDNA, irrespective of whether they encode apolypeptide having Neutrokine-α activity. This is because even where aparticular nucleic acid molecule does not encode a polypeptide havingNeutrokine-α activity, one of skill in the art would still know how touse the nucleic acid molecule, for instance, as a hybridization probe ora polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving Neutrokine-α activity include, inter alia, (1) isolating theNeutrokine-α gene or allelic variants thereof in a cDNA library; (2) insitu hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the Neutrokine-α gene, asdescribed in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and Northern Blot analysisfor detecting Neutrokine-α mRNA expression in specific tissues.

[0069] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIGS. 1A and B (SEQ ID NO:1) or to the nucleic acidsequence of the deposited cDNA which do, in fact, encode a polypeptidehaving Neutrokine-α protein activity. By “a polypeptide havingNeutrokine-α activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of theextracellular domain or of the full-length Neutrokine-α protein of theinvention, as measured in a particular biological assay. For example,the Neutrokine-α protein of the present invention modulates cellproliferation, cytotoxicity and cell death. An in vitro cellproliferation, cytotoxicity and cell death assay for measuring theeffect of a protein on certain cells can be performed by using reagentswell known and commonly available in the art for detecting cellreplication and/or death. For instance, numerous such assays forTNF-related protein activities are described in the various referencesin the Background section of this disclosure, above. Briefly, such anassay involves collecting human or animal (e.g., mouse) cells and mixingwith (1) transfected host cell-supernatant containing Neutrokine-αprotein (or a candidate polypeptide) or (2) nontransfected hostcell-supernatant control, and measuring the effect on cell numbers orviability after incubation of certain period of time. Such cellproliferation modulation activities as can be measure in this type ofassay are useful for treating tumor, tumor metastasis, infections,autoimmune diseases inflammation and other immune-related diseases.

[0070] Neutrokine-α modulates cell proliferation and differentiation ina dose-dependent manner in the above-described assay. Thus, “apolypeptide having Neutrokine a protein activity” includes polypeptidesthat also exhibit any of the same cell modulatory (particularlyimmunomodulatory) activities in the above-described assays in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the Neutrokine-α protein, preferably,“a polypeptide having Neutrokine-α protein activity” will exhibitsubstantially similar dose-dependence in a given activity as compared tothe Neutrokine-α protein (i.e., the candidate polypeptide will exhibitgreater activity or not more than about 25-fold less and, preferably,not more than about tenfold less activity relative to the referenceNeutrokine-α protein).

[0071] Like other members of TNF family, Neutrokine-α exhibits activityon leukocytes including for example monocytes, lymphocytes andneutrophils. For this reason Neutrokine-α is active in directing theproliferation, differentiation and migration of these cell types. Suchactivity is useful for immune enhancement or suppression,myeloprotection, stem cell mobilization, acute and chronic inflammatorycontrol and treatment of leukemia. Assays for measuring such activityare known in the art. For example, see Peters et al., Immun. Today17:273 (1996); Young et al., J. Exp. Med. 182:1111 (1995); Caux et al.,Nature 390:258 (1992); and Santiago-Schwarz et al., Adv. Exp. Med. Biol.378:7 (1995).”

[0072] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) willencode a polypeptide “having Neutrokine-α protein activity.” In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving Neutrokine-α protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid),as further described below. A further nucleic acid embodiment of theinvention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of a Neutrokine-αpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a polynucleotide which encodes the amino acid sequence of aNeutrokine-α polypeptide to have an amino acid sequence which containsnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acidsubstitutions.

[0073] Vectors and Host Cells

[0074] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof Neutrokine-α polypeptides or fragments thereof by recombinanttechniques. The vector may be, for example, a phage, plasmid, viral orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells. The polynucleotides may bejoined to a vector containing a selectable marker for propagation in ahost. Generally, a plasmid vector is introduced in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. If the vector is a virus, it may be packaged in vitro using anappropriate packaging cell line and then transduced into host cells.

[0075] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the extracellular domain of the transcripts expressed by theconstructs will preferably include a translation initiating at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

[0076] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0077] Among vectors preferred for use in bacteria include pHE4-5 (ATCCAccession No. 209311; and variations thereof), pQE70, pQE60 and pQE-9,available from QIAGEN, Inc., supra; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0078] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0079] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5 has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

[0080] The Neutrokine-α protein can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0081] Neutrokine-α Polypeptides and Fragments

[0082] The invention further provides an isolated Neutrokine-αpolypeptide having the amino acid sequence encoded by the depositedcDNA, or the amino acid sequence in FIGS. 1A and B (SEQ ID NO:2), or apeptide or polypeptide comprising a portion of the above polypeptides.

[0083] Variant and Mutant Polypeptides

[0084] To improve or alter the characteristics of Neutrokine-αpolypeptides, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or “muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

[0085] N-Terminal and C-Terminal Deletion Mutants

[0086] For instance, for many proteins, including the extracellulardomain or the mature form(s) of a secreted protein, it is known in theart that one or more amino acids may be deleted from the N-terminus orC-terminus without substantial loss of biological function. Forinstance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reportedmodified KGF proteins that had heparin binding activity even if 3, 8, or27 amino-terminal amino acid residues were missing.

[0087] In the present case, since the protein of the invention is amember of the TNF polypeptide family, deletions of N-terminal aminoacids up to the Gly (G) residue at position 191 in FIGS. 1A and B (SEQID NO:2) may retain some biological activity such as cytotoxicity toappropriate target cells. Polypeptides having further N-terminaldeletions including the Gly (G) residue would not be expected to retainsuch biological activities because it is known that this residue inTNF-related polypeptides is in the beginning of the conserved domainrequired for biological activities. However, even if deletion of one ormore amino acids from the N-terminus of a protein results inmodification of loss of one or more biological functions of the protein,other biological activities may still be retained. Thus, the ability ofthe shortened protein to induce and/or bind to antibodies whichrecognize the complete or extracellular domain of the protein generallywill be retained when less than the majority of the residues of thecomplete or extracellular domain of the protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

[0088] Accordingly, the present invention further provides polypeptideshaving one or more residues from the amino terminus of the amino acidsequence of the Neutrokine-α shown in FIG. 1 (SEQ ID NO:2), up to theglycine residue at position 191 (Gly-191 residue from the aminoterminus), and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides having the aminoacid sequence of residues n-285 of SEQ ID NO:2, where n is an integer inthe range of 2-190 and 191 is the position of the first residue from theN-terminus of the complete Neutrokine-α polypeptide (shown in SEQ IDNO:2) believed to be required for activity of the Neutrokine-α protein.More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues 2-285, 3-285,4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285,13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285,22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285,31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285,40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285,49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285,58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285,67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285,76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285, 84-285,85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285,94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285,102-285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-285,110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285,118-285, 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285,126-285, 127-285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285,134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285,142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285, 149-285,150-285, 151-285, 152-285, 153-285, 154-285, 155-285, 156-285, 157-285,158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285,166-285, 167-285, 168-285, 169-285, 170-285, 171-285, 172-285, 173-285,174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-285,182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285,and 190-285 of SEQ ID NO:2. Polynucleotides encoding these polypeptidesalso are provided.

[0089] Similarly, many examples of biologically functional C-terminaldeletion muteins are known. For instance, Interferon gamma shows up toten times higher activities by deleting 8-10 amino acid residues fromthe carboxy terminus of the protein (Döbeli et al., J. Biotechnology7:199-216 (1988). Since the present protein is a member of the TNFpolypeptide family, deletions of C-terminal amino acids up to theleucine residue at position 284 are expected to retain most if not allbiological activity such as receptor binding and modulation of cellreplication. Polypeptides having deletions of up to about 10 additionalC-terminal residues (i.e., up to the glycine residue at position 273)also may retain some activity such as receptor binding, although suchpolypeptides would lack a portion of the conserved TNF domain beginningat about Leu-284. However, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened protein toinduce and/or bind to antibodies which recognize the complete or matureprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

[0090] Accordingly, the present invention further provides polypeptideshaving one or more residues from the carboxy terminus of the amino acidsequence of the Neutrokine-α shown in FIGS. 1A and B (SEQ ID NO:2), upto the glycine residue at position 274 (Gly-274) and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides having the amino acid sequence of residues 1-m ofthe amino acid sequence in SEQ ID NO:2, where m is any integer in therange of 274 to 284. More in particular, the invention providespolynucleotides encoding polypeptides having the amino acid sequence ofresidues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282,1-283 and 1-284 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides also are provided.

[0091] Also provided are polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n-m of SEQ ID NO:2, where n and mare integers as described above. Also included are a nucleotide sequenceencoding a polypeptide consisting of a portion of the completeNeutrokine-α amino acid sequence encoded by the cDNA clone contained inATCC No. 97768 deposited on Oct. 22, 1996 where this portion excludesfrom 1 to 190 amino acids from the amino terminus or from 1 to 11 aminoacids from the C-terminus of the complete amino acid sequence (or anycombination of these N-terminal and C-terminal deletions) encoded by thecDNA clone in the deposited clone. Polynucleotides encoding all of theabove deletion polypeptides also are provided.

[0092] Other Mutants

[0093] In addition to terminal deletion forms of the protein discussedabove, it will be recognized by one of ordinary skill in the art thatsome amino acid sequences of the Neutrokine-α polypeptide can be variedwithout significant effect of the structure or function of the protein.If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity.

[0094] Thus, the invention further includes variations of theNeutrokine-α polypeptide which show substantial Neutrokine-α polypeptideactivity or which include regions of Neutrokine-α protein such as theprotein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions selectedaccording to general rules known in the art so as have little effect onactivity. For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main approaches for studying the toleranceof an amino acid sequence to change. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.

[0095] As the authors state, these studies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive ata certain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0096] Thus, the fragment, derivative or analog of the polypeptide ofFIGS. 1A and B (SEQ ID NO:2), or that encoded by the deposited cDNA, maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the extracellular domain of thepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe extracellular domain of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the extracellular domain of the polypeptideor a proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein

[0097] Thus, the Neutrokine-α of the present invention may include oneor more amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation. As indicated, changes arepreferably of a minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein (see Table 1). TABLE 1 Conservative Amino AcidSubstitutions. Aromatic Phenylalanine Tryptophan Tyrosine HydrophobicLeucine Isoleucine Valine Polar Glutamine Asparagine Basic ArginineLysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

[0098] Amino acids in the Neutrokine-α protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro or in vitroproliferative activity.

[0099] Of special interest are substitutions of charged amino acids withother charged or neutral amino acids which may produce proteins withhighly desirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

[0100] Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266-268 (1993) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Since Neutrokine-α is a member of the TNF polypeptide family,mutations similar to those in TNF-α are likely to have similar effectsin Neutrokine-α.

[0101] Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).Since Neutrokine-α is a member of the TNF-related protein family, tomodulate rather than completely eliminate biological activities ofNeutrokine-α, preferably mutations are made in sequences encoding aminoacids in the TNF conserved domain, i.e., in positions 191-284 of FIGS.1A and B (SEQ ID NO:2), more preferably in residues within this regionwhich are not conserved in all members of the TGF family. By making aspecific mutation in Neutrokine-α in the position where such a conservedamino acid is typically found in related TNFs, Neutrokine-α will act asan antagonist, thus possessing angiogenic activity. Accordingly,polypeptides of the present invention include Neutrokine-α mutants. SuchNeutrokine-α mutants are comprised of the full-length or preferably theextracellular domain of the Neutrokine-α amino acid sequence shown inFIGS. 1A and B (SEQ ID NO:2). Also forming part of the present inventionare isolated polynucleotides comprising nucleic acid sequences whichencode the above Neutrokine-α mutants.

[0102] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the Neutrokine-α polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0103] The polypeptides of the present invention include the completepolypeptide encoded by the deposited cDNA including the intracellular,transmembrane and extracellular domains of the polypeptide encoded bythe deposited cDNA, the extracellular domain minus the intracellular andtransmembrane domains of the protein, the complete polypeptide of FIGS.1A and B (SEQ ID NO:2), the extracellular domain of FIGS. 1A and B (SEQID NO:2) minus the intracellular and transmembrane domains, as well aspolypeptides which have at least 90% similarity, more preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above.

[0104] Further polypeptides of the present invention includepolypeptides at least 80% identical, more preferably at least 90% or 95%identical, still more preferably at least 96%, 97%, 98% or 99% identicalto the polypeptide encoded by the deposited cDNA or to the polypeptideof FIGS. 1A and B (SEQ ID NO:2), and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

[0105] A further embodiment of the invention relates to a peptide orpolypeptide which comprises the amino acid sequence of a Neutrokine-αpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a peptide or polypeptide to have an amino acid sequence whichcomprises the amino acid sequence of a Neutrokine-α polypeptide, whichcontains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

[0106] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics2:482-489, 1981) to find the best segment of similarity between twosequences.

[0107] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of aNeutrokine-α polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the Neutrokine-αpolypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0108] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2) or to theamino acid sequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0109] The polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0110] As described in detail below, the polypeptides of the presentinvention can also be used to raise polyclonal and monoclonalantibodies, which are useful in assays for detecting Neutrokine-αprotein expression as described below or as agonists and antagonistscapable of enhancing or inhibiting Neutrokine-α protein function.Further, such polypeptides can be used in the yeast two-hybrid system to“capture” Neutrokine-α protein binding proteins which are also candidateagonists and antagonists according to the present invention. The yeasttwo hybrid system is described in Fields and Song, Nature 340:245-246(1989).

[0111] Epitope-Bearing Portions

[0112] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0113] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) “Antibodiesthat react with predetermined sites on proteins”, Science, 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Antigenic epitope-bearingpeptides and polypeptides of the invention are therefore useful to raiseantibodies, including monoclonal antibodies, that bind specifically to apolypeptide of the invention. See, for instance, Wilson et al., Cell37:767-778 (1984) at 777.

[0114] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about 15 to about30 amino acids contained within the amino acid sequence of a polypeptideof the invention. Non-limiting examples of antigenic polypeptides orpeptides that can be used to generate Neutrokine-α specific antibodiesinclude: a polypeptide comprising amino acid residues from about Phe-115to about Leu-147 in FIGS. 1A and B (SEQ ID NO:2); a polypeptidecomprising amino acid residues from about Ile-150 to about Tyr-163 inFIGS. 1A and B (SEQ ID NO:2); a polypeptide comprising amino acidresidues from about Ser-171 to about Phe-194 in FIGS. 1A and B (SEQ IDNO:2); a polypeptide comprising amino acid residues from about Glu-223to about Tyr-247 in FIGS. 1A and B (SEQ ID NO:2); a polypeptidecomprising amino acid residues from about Ser-271 to about Phe-278 inFIGS. 1A and B (SEQ ID NO:2); These polypeptide fragments have beendetermined to bear antigenic epitopes of the Neutrokine-α protein by theanalysis of the Jameson-Wolf antigenic index, as shown in FIG. 3, above.

[0115] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. See, e.g., Houghten, R. A.(1985) General method for the rapid solid-phase synthesis of largenumbers of peptides: specificity of antigen-antibody interaction at thelevel of individual amino acids. Proc. Natl. Acad. Sci. USA82:5131-5135; this “Simultaneous Multiple Peptide Synthesis (SMPS)”process is further described in U.S. Pat. No. 4,631,211 to Houghten etal. (1986).

[0116] Epitope-bearing peptides and polypeptides of the invention areused to induce antibodies according to methods well known in the art.See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow,M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. etal., J. Gen. Virol. 66:2347-2354 (1985). Immunogenic epitope-bearingpeptides of the invention, i.e., those parts of a protein that elicit anantibody response when the whole protein is the immunogen, areidentified according to methods known in the art. See, for instance,Geysen et al., supra. Further still, U.S. Pat. No. 5,194,392 to Geysen(1990) describes a general method of detecting or determining thesequence of monomers (amino acids or other compounds) which is atopological equivalent of the epitope (i.e., a “mimotope”) which iscomplementary to a particular paratope (antigen binding site) of anantibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen(1989) describes a method of detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al.(1996) on Peralkylated Oligopeptide Mixtures discloses linearC1-C7-alkyl peralkylated oligopeptides and sets and libraries of suchpeptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

[0117] Fusion Proteins

[0118] As one of skill in the art will appreciate, Neutrokine-αpolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EP A 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric Neutrokine-α protein orprotein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)).

Immune System-Related Disorder Diagnosis

[0119] The present inventors have discovered that Neutrokine-α isexpressed in various tissues and particularly in neutrophils. For anumber of immune system-related disorders, substantially altered(increased or decreased) levels of Neutrokine-α gene expression can bedetected in immune system tissue or other cells or bodily fluids (e.g.,sera, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to a “standard” Neutrokine-αgene expression level, that is, the Neutrokine-α expression level inimmune system tissues or bodily fluids from an individual not having theimmune system disorder. Thus, the invention provides a diagnostic methoduseful during diagnosis of an system disorder, which involves measuringthe expression level of the gene encoding the Neutrokine-α protein inimmune system tissue or other cells or body fluid from an individual andcomparing the measured gene expression level with a standardNeutrokine-α gene expression level, whereby an increase or decrease inthe gene expression level compared to the standard is indicative of animmune system disorder.

[0120] In particular, it is believed that certain tissues in mammalswith cancer of the immune express significantly enhanced or reducedlevels of the Neutrokine-α protein and mRNA encoding the Neutrokine-αprotein when compared to a corresponding “standard” level. Further, itis believed that enhanced or depressed levels of the Neutrokine-αprotein can be detected in certain body fluids (e.g., sera, plasma,urine, and spinal fluid) from mammals with such a cancer when comparedto sera from mammals of the same species not having the cancer.

[0121] Thus, the invention provides a diagnostic method useful duringdiagnosis of a immune system disorder, including cancers of this system,which involves measuring the expression level of the gene encoding theNeutrokine-α protein in immune system tissue or other cells or bodyfluid from an individual and comparing the measured gene expressionlevel with a standard Neutrokine-α gene expression level, whereby anincrease or decrease in the gene expression level compared to thestandard is indicative of an immune system disorder.

[0122] Where a diagnosis of a disorder in the immune system, includingdiagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed Neutrokine-α geneexpression will experience a worse clinical outcome relative to patientsexpressing the gene at a level nearer the standard level.

[0123] By “assaying the expression level of the gene encoding theNeutrokine-α protein” is intended qualitatively or quantitativelymeasuring or estimating the level of the Neutrokine-α protein or thelevel of the mRNA encoding the Neutrokine-α protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the Neutrokine-α protein level or mRNA level in a second biologicalsample). Preferably, the Neutrokine-α protein level or mRNA level in thefirst biological sample is measured or estimated and compared to astandard Neutrokine-α protein level or mRNA level, the standard beingtaken from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having a disorder of the immune system. Aswill be appreciated in the art, once a standard Neutrokine-α proteinlevel or mRNA level is known, it can be used repeatedly as a standardfor comparison.

[0124] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains Neutrokine-α protein or mRNA. As indicated,biological samples include body fluids (such as sera, plasma, urine,synovial fluid and spinal fluid) which contain free extracellulardomains of the Neutrokine-α protein, immune system tissue, and othertissue sources found to express complete or free extracellular domain ofthe Neutrokine-α or a Neutrokine-α receptor. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

[0125] The present invention is useful for diagnosis or treatment ofvarious immune system-related disorders in mammals, preferably humans.Such disorders include but are not limited to tumors and tumormetastasis, infections by bacteria, viruses and other parasites,immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmunediseases, and graft versus host disease.

[0126] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the Neutrokine-α protein are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

[0127] Assaying Neutrokine-α protein levels in a biological sample canoccur using antibody-based techniques. For example, Neutrokine-α proteinexpression in tissues can be studied with classical immunohistologicalmethods (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985);Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Otherantibody-based methods useful for detecting Neutrokine α protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc),and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0128] In addition to assaying Neutrokine-α protein levels in abiological sample obtained from an individual, Neutrokine-α protein canalso be detected in vivo by imaging. Antibody labels or markers for invivo imaging of Neutrokine-α protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0129] A Neutrokine-α protein-specific antibody or antibody fragmentwhich has been labeled with an appropriate detectable imaging moiety,such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), aradio-opaque substance, or a material detectable by nuclear magneticresonance, is introduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain Neutrokine-α protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

[0130] Antibodies

[0131] Neutrokine-α-protein specific antibodies for use in the presentinvention can be raised against the intact Neutrokine-α protein or anantigenic polypeptide fragment thereof, which may be presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

[0132] As used herein, the term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as antibody fragments(such as, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to Neutrokine-α protein. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred.

[0133] The antibodies of the present invention may be prepared by any ofa variety of methods. For example, cells expressing the Neutrokine-αprotein or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of Neutrokine-α proteinis prepared and purified to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

[0134] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or Neutrokine-α protein bindingfragments thereof). Such monoclonal antibodies can be prepared usinghybridoma technology (Köhler et al., Nature 256:495 (1975); Köhler etal., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol.6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., (1981) pp. 563-681). In general, suchprocedures involve immunizing an animal (preferably a mouse) with aNeutrokine-α protein antigen or, more preferably, with a Neutrokine-αprotein-expressing cell. Suitable cells can be recognized by theircapacity to bind anti-Neutrokine-α protein antibody. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP2O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the Neutrokine-α protein antigen.

[0135] Alternatively, additional antibodies capable of binding to theNeutrokine-α protein antigen may be produced in a two-step procedurethrough the use of anti-idiotypic antibodies. Such a method makes use ofthe fact that antibodies are themselves antigens, and that, therefore,it is possible to obtain an antibody which binds to a second antibody.In accordance with this method, Neutrokine-α-protein specific antibodiesare used to immunize an animal, preferably a mouse. The splenocytes ofsuch an animal are then used to produce hybridoma cells, and thehybridoma cells are screened to identify clones which produce anantibody whose ability to bind to the Neutrokine-α protein-specificantibody can be blocked by the Neutrokine-α protein antigen. Suchantibodies comprise anti-idiotypic antibodies to the Neutrokine-αprotein-specific antibody and can be used to immunize an animal toinduce formation of further Neutrokine-α protein-specific antibodies.

[0136] It will be appreciated that Fab and F(ab′)2 and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). Alternatively,Neutrokine-α protein-binding fragments can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

[0137] Where in vivo imaging is used to detect enhanced levels ofNeutrokine-α protein for diagnosis in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

Treatment of Immune System-Related Disorders

[0138] As noted above, Neutrokine-α polynucleotides and polypeptides areuseful for diagnosis of conditions involving abnormally high or lowexpression of Neutrokine-α activities. Given the cells and tissues whereNeutrokine-α is expressed as well as the activities modulated byNeutrokine-α, it is readily apparent that a substantially altered(increased or decreased) level of expression of Neutrokine-α in anindividual compared to the standard or “normal” level producespathological conditions related to the bodily system(s) in whichNeutrokine-α is expressed and/or is active.

[0139] It will also be appreciated by one of ordinary skill that, sincethe Neutrokine-α protein of the invention is a member of the TNF family,the extracellular domain of the protein may be released in soluble formfrom the cells which express Neutrokine-α by proteolytic cleavage andtherefore, when Neutrokine-α protein (particularly a soluble form of theextracellular domain) is added from an exogenous source to cells,tissues or the body of an individual, the protein will exert itsmodulating activities on any of its target cells of that individual.Also, cells expressing this type II transmembrane protein may be addedto cells, tissues or the body of an individual whereby the added cellswill bind to cells expressing receptor for Neutrokine-α whereby thecells expressing Neutrokine-α can cause actions (e.g., cytotoxicity) onthe receptor-bearing target cells.

[0140] Therefore, it will be appreciated that conditions caused by adecrease in the standard or normal level of Neutrokine-α activity in anindividual, particularly disorders of the immune system, can be treatedby administration of Neutrokine-α protein (in the form of solubleextracellular domain or cells expressing the complete protein). Thus,the invention also provides a method of treatment of an individual inneed of an increased level of Neutrokine-α activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated Neutrokine-α polypeptide of theinvention, effective to increase the Neutrokine-α activity level in suchan individual.

[0141] Since Neutrokine-α belongs to the TNF superfamily, it also shouldalso modulate angiogenesis. In addition, since Neutrokine-α inhibitsimmune cell functions, it will have a wide range of anti-inflammatoryactivities. Neutrokine-α may be employed as an anti-neovascularizingagent to treat solid tumors by stimulating the invasion and activationof host defense cells, e.g., cytotoxic T cells and macrophages and byinhibiting the angiogenesis of tumors. Those of skill in the art willrecognize other non-cancer indications where blood vessel proliferationis not wanted. They may also be employed to enhance host defensesagainst resistant chronic and acute infections, for example,myobacterial infections via the attraction and activation ofmicrobicidal leukocytes. Neutrokine-α may also be employed to inhibitT-cell proliferation by the inhibition of IL-2 biosynthesis for thetreatment of T-cell mediated auto-immune diseases and lymphocyticleukemias. Neutrokine-α may also be employed to stimulate wound healing,both via the recruitment of debris clearing and connective tissuepromoting inflammatory cells. In this same manner, Neutrokine-α may alsobe employed to treat other fibrotic disorders, including livercirrhosis, osteoarthritis and pulmonary fibrosis. Neutrokine-α alsoincreases the presence of eosinophils which have the distinctivefunction of killing the larvae of parasites that invade tissues, as inschistosomiasis, trichinosis and ascariasis. It may also be employed toregulate hematopoiesis, by regulating the activation and differentiationof various hematopoietic progenitor cells, for example, to releasemature leukocytes from the bone marrow following chemotherapy, i.e., instem cell mobilization. Neutrokine-α may also be employed to treatsepsis.

[0142] Formulations

[0143] The Neutrokine-α polypeptide composition (preferably containing apolypeptide which is a soluble form of the extracellular domain) will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with Neutrokine-α polypeptidealone), the site of delivery of the Neutrokine-α polypeptidecomposition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of Neutrokine-α polypeptide for purposes herein is thusdetermined by such considerations.

[0144] As a general proposition, the total pharmaceutically effectiveamount of Neutrokine-α polypeptide administered parenterally per dosewill be in the range of about 1 μg/kg/day to 10 mg/kg/day of patientbody weight, although, as noted above, this will be subject totherapeutic discretion. More preferably, this dose is at least 0.01mg/kg/day, and most preferably for humans between about 0.01 and 1mg/kg/day for the hormone. If given continuously, the Neutrokine-αpolypeptide is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

[0145] Pharmaceutical compositions containing the Neutrokine-α of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0146] The Neutrokine-α polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release Neutrokine-α polypeptide compositions also includeliposomally entrapped Neutrokine-α polypeptide. Liposomes containingNeutrokine-α polypeptide are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal Neutrokine-α polypeptide therapy.

[0147] For parenteral administration, in one embodiment, theNeutrokine-α polypeptide is formulated generally by mixing it at thedesired degree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious topolypeptides.

[0148] Generally, the formulations are prepared by contacting theNeutrokine α polypeptide uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, more preferably a solution that is isotonicwith the blood of the recipient. Examples of such carrier vehiclesinclude water, saline, Ringer's solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes.

[0149] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0150] The Neutrokine-α polypeptide is typically formulated in suchvehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the useof certain of the foregoing excipients, carriers, or stabilizers willresult in the formation of Neutrokine-α polypeptide salts.

[0151] Neutrokine-α polypeptide to be used for therapeuticadministration must be sterile. Sterility is readily accomplished byfiltration through sterile filtration membranes (e.g., 0.2 micronmembranes). Therapeutic Neutrokine-α polypeptide compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0152] Neutrokine-α polypeptide ordinarily will be stored in unit ormulti-dose containers, for example, sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-ml vials are filled with 5ml of sterile-filtered 1% (w/v) aqueous Neutrokine-α polypeptidesolution, and the resulting mixture is lyophilized. The infusionsolution is prepared by reconstituting the lyophilized Neutrokine-αpolypeptide using bacteriostatic Water-for-Injection.

[0153] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

[0154] Agonists and Antagonists—Assays and Molecules

[0155] The invention also provides a method of screening compounds toidentify those which enhance or block the action of Neutrokine-α oncells, such as its interaction with Neutrokine-α binding molecules suchas receptor molecules. An agonist is a compound which increases thenatural biological functions of Neutrokine-α or which functions in amanner similar to Neutrokine while antagonists decrease or eliminatesuch functions.

[0156] In another aspect of this embodiment the invention provides amethod for identifying a receptor protein or other ligand-bindingprotein which binds specifically to a Neutrokine-α polypeptide. Forexample, a cellular compartment, such as a membrane or a preparationthereof, may be prepared from a cell that expresses a molecule thatbinds Neutrokine-α. The preparation is incubated with labeledNeutrokine-α and complexes of Neutrokine-α bound to the receptor orother binding protein are isolated and characterized according toroutine methods known in the art. Alternatively, the Neutrokine-αpolypeptide may be bound to a solid support so that binding moleculessolubilized from cells are bound to the column and then eluted andcharacterized according to routine methods.

[0157] In the assay of the invention for agonists or antagonists, acellular compartment, such as a membrane or a preparation thereof, maybe prepared from a cell that expresses a molecule that bindsNeutrokine-α such as a molecule of a signaling or regulatory pathwaymodulated by Neutrokine-α. The preparation is incubated with labeledNeutrokine-α in the absence or the presence of a candidate moleculewhich may be a Neutrokine-α agonist or antagonist. The ability of thecandidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Molecules which bindgratuitously, i.e., without inducing the effects of Neutrokine-α onbinding the Neutrokine-α binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to Neutrokine-α are agonists.

[0158] Neutrokine-α-like effects of potential agonists and antagonistsmay by measured, for instance, by determining activity of a secondmessenger system following interaction of the candidate molecule with acell or appropriate cell preparation, and comparing the effect with thatof Neutrokine-α or molecules that elicit the same effects asNeutrokine-α. Second messenger systems that may be useful in this regardinclude but are not limited to AMP guanylate cyclase, ion channel orphosphoinositide hydrolysis second messenger systems.

[0159] Another example of an assay for Neutrokine-α antagonists is acompetitive assay that combines Neutrokine-α and a potential antagonistwith membrane-bound receptor molecules or recombinant Neutrokine-αreceptor molecules under appropriate conditions for a competitiveinhibition assay. Neutrokine-α can be labeled, such as by radioactivity,such that the number of Neutrokine-α molecules bound to a receptormolecule can be determined accurately to assess the effectiveness of thepotential antagonist.

[0160] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingNeutrokine-α induced activities, thereby preventing the action ofNeutrokine-α by excluding Neutrokine-α from binding.

[0161] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano, J. Neurochem. 56: 560(1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the extracellular domain of thepolypeptide of the present invention may be used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription thereby preventing transcription and theproduction of Neutrokine-α. The antisense RNA oligonucleotide hybridizesto the mRNA in vivo and blocks translation of the mRNA molecule intoNeutrokine-α polypeptide. The oligonucleotides described above can alsobe delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of Neutrokine-α.

[0162] The agonists and antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as described above.

[0163] The antagonists may be employed for instance to inhibitNeutrokine-α the chemotaxis and activation of macrophages and theirprecursors, and of neutrophils, basophils, B lymphocytes and some T-cellsubsets, e.g., activated and CD8 cytotoxic T cells and natural killercells, in certain auto-immune and chronic inflammatory and infectivediseases. Examples of auto-immune diseases include multiple sclerosis,and insulin-dependent diabetes. The antagonists may also be employed totreat infectious diseases including silicosis, sarcoidosis, idiopathicpulmonary fibrosis by preventing the recruitment and activation ofmononuclear phagocytes. They may also be employed to treat idiopathichyper-eosinophilic syndrome by preventing eosinophil production andmigration. Endotoxic shock may also be treated by the antagonists bypreventing the migration of macrophages and their production of thehuman chemokine polypeptides of the present invention. The antagonistsmay also be employed for treating atherosclerosis, by preventingmonocyte infiltration in the artery wall. The antagonists may also beemployed to treat histamine-mediated allergic reactions andimmunological disorders including late phase allergic reactions, chronicurticaria, and atopic dermatitis by inhibiting chemokine-induced mastcell and basophil degranulation and release of histamine. IgE-mediatedallergic reactions such as allergic asthma, rhinitis, and eczema mayalso be treated. The antagonists may also be employed to treat chronicand acute inflammation by preventing the attraction of monocytes to awound area. They may also be employed to regulate normal pulmonarymacrophage populations, since chronic and acute inflammatory pulmonarydiseases are associated with sequestration of mononuclear phagocytes inthe lung. Antagonists may also be employed to treat rheumatoid arthritisby preventing the attraction of monocytes into synovial fluid in thejoints of patients. Monocyte influx and activation plays a significantrole in the pathogenesis of both degenerative and inflammatoryarthropathies. The antagonists may be employed to interfere with thedeleterious cascades attributed primarily to IL-1 and TNF, whichprevents the biosynthesis of other inflammatory cytokines. In this way,the antagonists may be employed to prevent inflammation. The antagonistsmay also be employed to inhibit prostaglandin-independent fever inducedby chemokines. The antagonists may also be employed to treat cases ofbone marrow failure, for example, aplastic anemia and myelodysplasticsyndrome. The antagonists may also be employed to treat asthma andallergy by preventing eosinophil accumulation in the lung. Theantagonists may also be employed to treat subepithelial basementmembrane fibrosis which is a prominent feature of the asthmatic lung.

[0164] Antibodies against Neutrokine-α may be employed to bind to andinhibit Neutrokine-α activity to treat ARDS, by preventing infiltrationof neutrophils into the lung after injury. The antagonists may beemployed in a composition with a pharmaceutically acceptable carrier,e.g., as hereinafter described.

[0165] Chromosome Assays

[0166] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0167] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a Neutrokine-α protein gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

[0168] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Fluorescence in situhybridization (“FISH”) of a cDNA clone to a metaphase chromosomal spreadcan be used to provide a precise chromosomal location in one step. Thistechnique can be used with probes from the cDNA as short as 50 or 60 bp.For a review of this technique, see Verma et al., Human Chromosomes: AManual Of Basic Techniques, Pergamon Press, New York (1988).

[0169] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance In Man, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0170] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0171] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1a Expression and Purification of “His-Tagged”Neutrokine-α in E. coli

[0172] The bacterial expression vector pQE9 (PD10) is used for bacterialexpression in this example. (QIAGEN, Inc., supra). pQE9 encodesampicillin antibiotic resistance (“Ampr”) and contains a bacterialorigin of replication (“ori”), an IPTG inducible promoter, a ribosomebinding site (“RBS”), six codons encoding histidine residues that allowaffinity purification using nickel-nitrilo-tri-acetic acid (“Ni-NTA”)affinity resin sold by QIAGEN, Inc., supra, and suitable singlerestriction enzyme cleavage sites. These elements are arranged such thatan inserted DNA fragment encoding a polypeptide expresses thatpolypeptide with the six His residues (i.e., a “6× His tag”) covalentlylinked to the amino terminus of that polypeptide.

[0173] The DNA sequence encoding the desired portion of the Neurokine-αprotein comprising the extracellular domain sequence is amplified fromthe deposited cDNA clone using PCR oligonucleotide primers which annealto the amino terminal sequences of the desired portion of theNeurokine-α protein and to sequences in the deposited construct 3′ tothe cDNA coding sequence. Additional nucleotides containing restrictionsites to facilitate cloning in the pQE9 vector are added to the 5′ and3′ primer sequences, respectively.

[0174] For cloning the extracellular domain of the protein, the 5′primer has the sequence 5′ GTG GGA TCC AGC CTC CGG GCA GAG CTG 3′ (SEQID NO:10) containing the underlined Bam HI restriction site followed by18 nucleotides of the amino terminal coding sequence of theextracellular domain of the Neurokine-α sequence in FIGS. 1A and B. Oneof ordinary skill in the art would appreciate, of course, that the pointin the protein coding sequence where the 5′ primer begins may be variedto amplify a DNA segment encoding any desired portion of the completeNeutrokine α protein shorter or longer than the extracellular domain ofthe form. The 3′ primer has the sequence 5′-GTG AAG CTT TTA TTA CAG CAGTTT CAA TGC ACC-3′ (SEQ ID NO:11) containing the underlined Hind IIIrestriction site followed by two stop codons and 18 nucleotidescomplementary to the 3′ end of the coding sequence of the Neurokine-αDNA sequence in FIGS. 1A and B.

[0175] The amplified Neurokine-α DNA fragment and the vector pQE9 aredigested with Bam HI and Hind III and the digested DNAs are then ligatedtogether. Insertion of the Neurokine-α DNA into the restricted pQE9vector places the Neurokine-α protein coding region downstream from theIPTG-inducible promoter and in-frame with an initiating AUG and the sixhistidine codons.

[0176] The ligation mixture is transformed into competent E. coli cellsusing standard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing Neurokine-α protein, is available commercially from QIAGEN,Inc., supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing. Clonescontaining the desired constructs are grown overnight (“O/N”) in liquidculture in LB media supplemented with both ampicillin (100 μg/ml) andkanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

[0177] The cells are then stirred for 3-4 hours at 4° C. in 6Mguanidine-HCl, pH 8. The cell debris is removed by centrifugation, andthe supernatant containing the Neurokine-α is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist, 1995, QIAGEN,Inc., supra). Briefly the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the Neurokine-α is eluted with 6 M guanidine-HCl, pH 5.

[0178] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

Example 1b Expression and Purification of Neutrokine-α in E. coli

[0179] The bacterial expression vector pQE60 is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr”)and contains a bacterial origin of replication (“ori”), an IPTGinducible promoter, a ribosome binding site (“RBS”), six codons encodinghistidine residues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6× His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6× His tag.

[0180] The DNA sequence encoding the desired portion of the Neurokine-αprotein comprising the extracellular domain sequence is amplified fromthe deposited cDNA clone using PCR oligonucleotide primers which annealto the amino terminal sequences of the desired portion of theNeurokine-α protein and to sequences in the deposited construct 3′ tothe cDNA coding sequence. Additional nucleotides containing restrictionsites to facilitate cloning in the pQE60 vector are added to the 5′ and3′ sequences, respectively.

[0181] For cloning the extracellular domain of the protein, the 5′primer has the sequence 5′ GTG TCA TGA GCC TCC GGG CAG AGC TG 3′ (SEQ IDNO:12) containing the underlined Bsp HI restriction site followed by 17nucleotides of the amino terminal coding sequence of the extracellulardomain of the Neurokine-α sequence in FIGS. 1A and B. One of ordinaryskill in the art would appreciate, of course, that the point in theprotein coding sequence where the 5′ primer begins may be varied toamplify a desired portion of the complete protein shorter or longer thanthe extracellular domain of the form. The 3′ primer has the sequence5′-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC ACC 3′ (SEQ 13) containingthe underlined Hind III restriction site followed by two stop codons 8nucleotides complementary to the 3′ end of the coding sequence in theNeurokine-α DNA sequence in FIGS. 1A and B.

[0182] The amplified Neurokine-α DNA fragments and the vector pQE60 aredigested with Bsp HI and Hind III and the digested DNAs are then ligatedtogether. Insertion of the Neurokine-α DNA into the restricted pQE60vector places the Neurokine-α protein coding region including itsassociated stop codon downstream from the IPTG-inducible promoter andin-frame with an initiating AUG. The associated stop codon preventstranslation of the six histidine codons downstream of the insertionpoint.

[0183] The ligation mixture is transformed into competent E. coli cellsusing standard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingNeurokine-α protein, is available commercially from QIAGEN, Inc., supra.Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

[0184] One of ordinary skill in the art recognizes that any of a numberof bacterial expression vectors may be useful in place of pQE9 and pQE60in the expression protocols presented in this example. For example, thenovel pHE4 series of bacterial expression vectors, in particular, thepHE4-5 vector may be used for bacterial expression in this example (ATCCAccession No. 209311; and variations thereof). The plasmid DNAdesignated pHE4-5/MPIFΔ23 in ATCC Deposit No. 209311 is vector plasmidDNA which contains an insert which encodes another ORF. The constructwas deposited with the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852, on Sep. 30, 1997. Using the Nde I and Asp718 restriction sites flanking the irrelevant MPIF ORF insert, one ofordinary skill in the art could easily use current molecular biologicaltechniques to replace the irrelevant ORF in the pHE4-5 vector with theNeutrokine-α ORF of the present invention.

[0185] The pHE4-5 bacterial expression vector includes a neomycinphosphotransferase gene for selection, an E. coli origin of replication,a T5 phage promoter sequence, two lac operator sequences, aShine-Delgarno sequence, and the lactose operon repressor gene (lacIq).These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6× His tag”) covalently linked to the amino terminus of thatpolypeptide. The promoter and operator sequences of the pHE4-5 vectorwere made synthetically. Synthetic production of nucleic acid sequencesis well known in the art (CLONETECH 95/96 Catalog, pages 215-216,CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).

[0186] Clones containing the desired Neutrokine-α constructs are grownovernight (“O/N”) in liquid culture in LB media supplemented with bothampicillin (100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is usedto inoculate a large culture, at a dilution of approximately 1:25 to1:250. The cells are grown to an optical density at 600 nm (“OD600”) ofbetween 0.4 and 0.6. isopropyl-b-D-thiogalactopyranoside (“IPTG”) isthen added to a final concentration of 1 mM to induce transcription fromthe lac repressor sensitive promoter, by inactivating the lacIrepressor. Cells subsequently are incubated further for 3 to 4 hours.Cells then are harvested by centrifugation.

[0187] The cells are then stirred for 3-4 hours at 4° C. in 6Mguanidine-HCl, pH 8. The cell debris is removed by centrifugation, andthe supernatant containing the Neutrokine α is dialyzed against 50 mMNa-acetate buffer pH 6, supplemented with 200 mM NaCl. Alternatively,the protein can be successfully refolded by dialyzing it against 500 mMNaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4, containing proteaseinhibitors. After renaturation the protein can be purified by ionexchange, hydrophobic interaction and size exclusion chromatography.Alternatively, an affinity chromatography step such as an antibodycolumn can be used to obtain pure Neurokine-α protein. The purifiedprotein is stored at 4° C. or frozen at −80° C.

Example 2 Cloning and Expression of Neutrokine-α Protein in aBaculovirus Expression System

[0188] In this illustrative example, the plasmid shuttle vector pA2GP isused to insert the cloned DNA encoding the extracellular domain of theprotein, lacking its naturally associated intracellular andtransmembrane sequences, into a baculovirus to express the extracellulardomain of the Neurokine-α protein, using a baculovirus leader andstandard methods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe secretory signal peptide (leader) of the baculovirus gp67 proteinand convenient restriction sites such as Bam HI, Xba I and Asp 718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpresses the cloned polynucleotide.

[0189] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[0190] The cDNA sequence encoding the extracellular domain of theNeurokine-α protein in the deposited clone, lacking the AUG initiationcodon and the naturally associated intracellular and transmembranedomain sequences shown in FIGS. 1A and B (SEQ ID NO:2 is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene. The 5′ primer has the sequence 5′-GTG GGA TCC CCG GGC AGA GCTGCA GGG C-3′ (SEQ ID NO:14) containing the underlined Bam HI restrictionenzyme site followed by 18 nucleotides of the sequence of theextracellular domain of the Neurokine-α protein shown in FIGS. 1A and B,beginning with the indicated N-terminus of the extracellular domain ofthe protein. The 3′ primer has the sequence 5′-GTG GGA TCC TTA TTA CAGCAG TTT CAA TGC ACC-3′ (SEQ ID NO:15) containing the underlined Bam HIrestriction site followed by two stop codons and 18 nucleotidescomplementary to the 3′ coding sequence in FIGS. 1A and B.

[0191] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Bam HI and again is purifiedon a 1% agarose gel. This fragment is designated herein F1.

[0192] The plasmid is digested with the restriction enzymes Bam HI andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V1”.

[0193] Fragment F1 and the dephosphorylated plasmid V1 are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the humanNeurokine-α gene by digesting DNA from individual colonies using Bam HIand then analyzing the digestion product by gel electrophoresis. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pA2GP-Neutrokine-α.

[0194] Five μg of the plasmid pA2GP-Neutrokine-α is co-transfected with1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84: 7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μgof the plasmid pA2GP Neurokine-α are mixed in a sterile well of amicrotiter plate containing 50 μl of serum-free Grace's medium (LifeTechnologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus90 μl Grace's medium are added, mixed and incubated for 15 minutes atroom temperature. Then the transfection mixture is added drop-wise toSf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is then incubated for5 hours at 27° C. The transfection solution is then removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. Cultivation is then continued at 27° C. for four days.

[0195] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-Neurokine-α.

[0196] To verify the expression of the Neurokine-α gene Sf9 cells aregrown in Grace's medium supplemented with 10% heat-inactivated FBS. Thecells are infected with the recombinant baculovirus V-Neurokine-α at amultiplicity of infection (“MOI”) of about 2. If radiolabeled proteinsare desired, 6 hours later the medium is removed and is replaced withSF900 II medium minus methionine and cysteine (available from LifeTechnologies Inc., Rockville, Md.). After 42 hours, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0197] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the extracellular domain of the protein and thus the cleavage pointand length of the secretory signal peptide.

Example 3 Cloning and Expression of Neutrokine-α in Mammalian Cells

[0198] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0199] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

[0200] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0201] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites Bam HI, Xba I and Asp 718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0202] The expression plasmid, pNeurokine-α-HA, is made by cloning aportion of the deposited cDNA encoding the extracellular domain of theNeurokine-α protein into the expression vector pcDNAI/Amp or pcDNAIII(which can be obtained from Invitrogen, Inc.). To produce a soluble,secreted form of the polypeptide, the extracellular domain is fused tothe secretory leader sequence of the human IL-6 gene.

[0203] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37: 767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0204] A DNA fragment encoding the extracellular domain of theNeurokine-α polypeptide is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The plasmid construction strategy is as follows. TheNeurokine-α cDNA of the deposited clone is amplified using primers thatcontain convenient restriction sites, much as described above forconstruction of vectors for expression of Neurokine-α in E. coli.Suitable primers include the following, which are used in this example.The 5′ primer, containing the underlined Bam HI site, a Kozak sequence,an AUG start codon, a sequence encoding the secretory leader peptidefrom the human IL-6 gene, and 18 nucleotides of the 5′ coding region ofthe extracellular domain of Neurokine-α protein, has the followingsequence: 5′-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGTCCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCCCCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3′ (SEQ ID NO:16). The 3′primer, containing the underlined Bam HI restriction site and 18 ofnucleotides complementary to the 3′ coding sequence immediately beforethe stop codon, has the following sequence: 5′-GTG GGA TCC TTA CAG CAGTTT CAA TGC ACC-3′ (SEQ ID NO:17).

[0205] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with Bam HI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding theNeutrokine-α extracellular domain.

[0206] For expression of recombinant Neurokine-α, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof Neurokine-α by the vector.

[0207] Expression of the Neurokine-α-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

[0208] The vector pC4 is used for the expression of Neurokine-α protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). To produce a soluble, secreted form of the Neurokine-αpolypeptide, the portion of the deposited cDNA encoding theextracellular domain is fused to the secretory leader sequence of thehuman IL-6 gene. The vector plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary- or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990,Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A.1991, Biotechnology 9:64-68). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

[0209] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter are the followingsingle restriction enzyme cleavage sites that allow the integration ofthe genes: BamHI, Xba I, and Asp718. Behind these cloning sites theplasmid contains the 3′ intron and polyadenylation site of the ratpreproinsulin gene. Other high efficiency promoters can also be used forthe expression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the Neurokine-α in a regulatedway in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89: 5547-5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0210] The plasmid pC4 is digested with the restriction enzymes Bam HIand then dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0211] The DNA sequence encoding the extracellular domain of theNeutrokine-α protein is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene. The 5′ primer,containing the underlined Bam HI site, a Kozak sequence, an AUG startcodon, a sequence encoding the secretory leader peptide from the humanIL-6 gene, and 18 nucleotides of the 5′ coding region of theextracellular domain of Neurokine-α protein, has the following sequence:5′-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTTGCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTTGTG AGA CAA GGG GAC CTG GCC AGC-3′ (SEQ ID NO:16). The 3′ primer,containing the underlined Bam HI and 18 of nucleotides complementary tothe 3′ coding sequence immediately before the stop codon, has thefollowing sequence: 5′-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3′ (SEQID NO:17).

[0212] The amplified fragment is digested with the endonuclease Bam HIand then purified again on a 1% agarose gel. The isolated fragment andthe dephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

[0213] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of Neutrokine-α mRNA Expression

[0214] Northern blot analysis is carried out to examine Neutrokine-αgene expression in human tissues, using methods described by, amongothers, Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the Neutrokine-α protein (SEQ ID NO:1) is labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for Neutrokine-α mRNA.

[0215] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with the labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

[0216] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0217] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 17 1100 base pairs nucleic acid single linear DNA (genomic) CDS147..1001 sig_peptide 285..381 mat_peptide 147..1001 1 AAATTCAGGATAACTCTCCT GAGGGGTGAG CCAAGCCCTG CCATGTAGTG CACGCAGGAC 60 ATCAACAAACACAGATAACA GGAAATGATC CATTCCCTGT GGTCACTTAT TCTAAAGGCC 120 CCAACCTTCAAAGTTCAAGT AGTGAT ATG GAT GAC TCC ACA GAA AGG GAG CAG 173 Met Asp AspSer Thr Glu Arg Glu Gln 1 5 TCA CGC CTT ACT TCT TGC CTT AAG AAA AGA GAAGAA ATG AAA CTG AAG 221 Ser Arg Leu Thr Ser Cys Leu Lys Lys Arg Glu GluMet Lys Leu Lys 10 15 20 25 GAG TGT GTT TCC ATC CTC CCA CGG AAG GAA AGCCCC TCT GTC CGA TCC 269 Glu Cys Val Ser Ile Leu Pro Arg Lys Glu Ser ProSer Val Arg Ser 30 35 40 TCC AAA GAC GGA AAG CTG CTG GCT GCA ACC TTG CTGCTG GCA CTG CTG 317 Ser Lys Asp Gly Lys Leu Leu Ala Ala Thr Leu Leu LeuAla Leu Leu 45 50 55 TCT TGC TGC CTC ACG GTG GTG TCT TTC TAC CAG GTG GCCGCC CTG CAA 365 Ser Cys Cys Leu Thr Val Val Ser Phe Tyr Gln Val Ala AlaLeu Gln 60 65 70 GGG GAC CTG GCC AGC CTC CGG GCA GAG CTG CAG GGC CAC CACGCG GAG 413 Gly Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His AlaGlu 75 80 85 AAG CTG CCA GCA GGA GCA GGA GCC CCC AAG GCC GGC CTG GAG GAAGCT 461 Lys Leu Pro Ala Gly Ala Gly Ala Pro Lys Ala Gly Leu Glu Glu Ala90 95 100 105 CCA GCT GTC ACC GCG GGA CTG AAA ATC TTT GAA CCA CCA GCTCCA GGA 509 Pro Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala ProGly 110 115 120 GAA GGC AAC TCC AGT CAG AAC AGC AGA AAT AAG CGT GCC GTTCAG GGT 557 Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val GlnGly 125 130 135 CCA GAA GAA ACA GTC ACT CAA GAC TGC TTG CAA CTG ATT GCAGAC AGT 605 Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala AspSer 140 145 150 GAA ACA CCA ACT ATA CAA AAA GGA TCT TAC ACA TTT GTT CCATGG CTT 653 Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro TrpLeu 155 160 165 CTC AGC TTT AAA AGG GGA AGT GCC CTA GAA GAA AAA GAG AATAAA ATA 701 Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn LysIle 170 175 180 185 TTG GTC AAA GAA ACT GGT TAC TTT TTT ATA TAT GGT CAGGTT TTA TAT 749 Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln ValLeu Tyr 190 195 200 ACT GAT AAG ACC TAC GCC ATG GGA CAT CTA ATT CAG AGGAAG AAG GTC 797 Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg LysLys Val 205 210 215 CAT GTC TTT GGG GAT GAA TTG AGT CTG GTG ACT TTG TTTCGA TGT ATT 845 His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe ArgCys Ile 220 225 230 CAA AAT ATG CCT GAA ACA CTA CCC AAT AAT TCC TGC TATTCA GCT GGC 893 Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr SerAla Gly 235 240 245 ATT GCA AAA CTG GAA GAA GGA GAT GAA CTC CAA CTT GCAATA CCA AGA 941 Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala IlePro Arg 250 255 260 265 GAA AAT GCA CAA ATA TCA CTG GAT GGA GAT GTC ACATTT TTT GGT GCA 989 Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr PhePhe Gly Ala 270 275 280 TTG AAA CTG CTG TGACCTACTT ACACCATGTC TGTAGCTATTTTCCTCCCTT 1041 Leu Lys Leu Leu 285 TCTCTGTACC TCTAAGAAGA AAGAATCTAACTGAAAATAC CAAAAAAAAA AAAAAAAAA 1100 285 amino acids amino acid linearprotein 2 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser CysLeu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser IleLeu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly LysLeu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu ThrVal Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala SerLeu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro AlaGly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val ThrAla Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly AsnSer Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro GluGlu Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser GluThr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro TrpLeu Leu Ser Phe Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys Glu AsnLys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile Tyr Gly GlnVal Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly His Leu Ile GlnArg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val ThrLeu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro AsnAsn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255 AspGlu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 285 233amino acids amino acid single linear protein 3 Met Ser Thr Glu Ser MetIle Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys ThrGly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe SerPhe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His PheGly Val Ile Gly Pro Gln Arg Glu Glu Ser Pro 50 55 60 Arg Asp Leu Ser LeuIle Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr ProSer Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu GlyGln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala AsnGly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu GlyLeu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 CysPro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp ArgLeu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala GluSer Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 205amino acids amino acid single linear protein 4 Met Thr Pro Pro Glu ArgLeu Phe Leu Pro Arg Val Cys Gly Thr Thr 1 5 10 15 Leu His Leu Leu LeuLeu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30 Gln Gly Leu Pro GlyVal Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45 Arg Gln His Pro LysMet His Leu Ala His Ser Thr Leu Lys Pro Ala 50 55 60 Ala His Leu Ile GlyAsp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg 65 70 75 80 Ala Asn Thr AspArg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95 Asn Ser Leu LeuVal Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105 110 Val Val PheSer Gly Lys Ala Tyr Ser Pro Lys Ala Pro Ser Ser Pro 115 120 125 Leu TyrLeu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe 130 135 140 HisVal Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln 145 150 155160 Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr 165170 175 Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val180 185 190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 195 200205 244 amino acids amino acid single linear protein 5 Met Gly Ala LeuGly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg 1 5 10 15 Gly Ser LeuLeu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu 20 25 30 Leu Leu AlaVal Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro 35 40 45 Gln Asp GlnGly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln 50 55 60 Ala Gln GlnGly Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu 65 70 75 80 Thr AspLeu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro 85 90 95 Leu LysGly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe 100 105 110 LeuThr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro 115 120 125Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg 130 135140 Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg 145150 155 160 Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr ProGlu 165 170 175 Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu AspPro Ala 180 185 190 Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser ValGly Phe Gly 195 200 205 Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val TyrVal Asn Ile Ser 210 215 220 His Pro Asp Met Val Asp Phe Ala Arg Gly LysThr Phe Phe Gly Ala 225 230 235 240 Val Met Val Gly 281 amino acidsamino acid single linear protein 6 Met Gln Gln Pro Phe Asn Tyr Pro TyrPro Gln Ile Tyr Trp Val Asp 1 5 10 15 Ser Ser Ala Ser Ser Pro Trp AlaPro Pro Gly Thr Val Leu Pro Cys 20 25 30 Pro Thr Ser Val Pro Arg Arg ProGly Gln Arg Arg Pro Pro Pro Pro 35 40 45 Pro Pro Pro Pro Pro Leu Pro ProPro Pro Pro Pro Pro Pro Leu Pro 50 55 60 Pro Leu Pro Leu Pro Pro Leu LysLys Arg Gly Asn His Ser Thr Gly 65 70 75 80 Leu Cys Leu Leu Val Met PhePhe Met Val Leu Val Ala Leu Val Gly 85 90 95 Leu Gly Leu Gly Met Phe GlnLeu Phe His Leu Gln Lys Glu Leu Ala 100 105 110 Glu Leu Arg Glu Ser ThrSer Gln Met His Thr Ala Ser Ser Leu Glu 115 120 125 Lys Gln Ile Gly HisPro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg 130 135 140 Lys Val Ala HisLeu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu 145 150 155 160 Glu TrpGlu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr 165 170 175 LysLys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr 180 185 190Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser 195 200205 His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met 210215 220 Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala225 230 235 240 Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser AlaAsp His 245 250 255 Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn PheGlu Glu Ser 260 265 270 Gln Thr Phe Phe Gly Leu Tyr Lys Leu 275 280 338base pairs nucleic acid single linear DNA (genomic) 7 AGGNTAACTCTCCTGAGGGG TGAGCCAAGC CCTGCCATGT AGTGCACGCA GGACATCANC 60 AAACACANNNNNCAGGAAAT AATCCATTCC CTGTGGTCAC TTATTCTAAA GGCCCCAACC 120 TTCAAAGTTCAAGTAGTGAT ATGGATGACT CCACAGAAAG GGAGCAGTCA CGCCTTACTT 180 CTTGCCTTAAGAAAAGAGAA GAAATGAAAC TGNAAGGAGT GTGTTTCCAT CCTCCCACGG 240 AAGGAAAGCCCCTCTNTCCG ATCCTCCAAA GACGGAAAGC TGCTGGCTGC AACCTTGNTG 300 NTGGCATTGTGTTCTTGCTG NCTCAAGGTG GTGTTNTT 338 509 base pairs nucleic acid singlelinear DNA (genomic) 8 AATTCGGCAN AGNAAACTGG TTACTTTTTT ATATATGGTCAGGTTTTATA TACTGATAAG 60 ACCTACGCCA TGGGACATCT AGTTCAGAGG AAGAAGGTCCATGTCTTTGG GGATGAATTG 120 AGTCTGGTGA CTTTGTTTCG ATGTATTCAA AATATGCCTGAAACACTACC CAATAATTCC 180 TGCTATTCAG CTGGCATTGC AAAACTGGNA GGAAGGAGATGAACTCCAAC TTGCAATACC 240 AGGGGAAAAT GCACAATTAT CACTGGGATG GAGATGTTCACATTTTTTGG GTGCCATTGA 300 AACTGCTGTG ACCTNCTTAC ANCANGTGCT GTTNGCTATTTTNCCTNCCT NTTCTNTGGT 360 AACCTCTTAG GAAGGAAGGA TTCTTAACTG GGAAATAACCCAAAAAAANN TTAAANGGGT 420 ANGNGNNANA NGNGGGGNNG TTNNCNNGNN GNNTTTTNGGNNTATNTTNT NNTNGGGNNN 480 NGTAAAAATG GGGCCNANGG GGGNTTTTT 509 497 basepairs nucleic acid single linear DNA (genomic) 9 AATTCGGCAC GAGCAAGGCCGGCCTGGAGG AAGCTCCAGC TGTCACCGCG GGACTGAAAA 60 TCTTTGAACC ACCAGCTCCAGGAGAAGGCA ACTCCAGTCA GAACAGCAGA AATAAGCGTG 120 CCGTTCAGGG TCCAGAAGAAACAGTCACTC AAGACTGCTT GCAACTGNTT GCAGACAGTG 180 AAACACCAAC TATACAAAAAGGCTCCCTTC TGNTGCCACA TTTGGGCCAA GGAATGGAGA 240 GATTTCTTCG TCTGGAAACATTTTGCCAAA CTCTTCAGAT ACTCTTTNCT CTCTGGGAAT 300 CAAAGGAAAA TCTCTACTTAGATTNACACA TTTGTTCCCA TGGGTNTCTT AAGTTTTAAA 360 AGGGGAGTGC CCTTAGGAGGAAAAGGGGAT AAATATTGGC CAAGGNACTG GTTANTTTNT 420 AAATATGGTC AGGTTTNTATANCTGGTAGG CCTCGCCATG GGCATTNATT CANGGNGAGG 480 NCNNTCTTTT GGGNTGA 49727 base pairs nucleic acid single linear DNA (genomic) 10 GTGGGATCCAGCCTCCGGGC AGAGCTG 27 33 base pairs nucleic acid single linear DNA(genomic) 11 GTGAAGCTTT TATTACAGCA GTTTCAATGC ACC 33 26 base pairsnucleic acid single linear DNA (genomic) 12 GTGTCATGAG CCTCCGGGCA GAGCTG26 33 base pairs nucleic acid single linear DNA (genomic) 13 GTGAAGCTTTTATTACAGCA GTTTCAATGC ACC 33 28 base pairs nucleic acid single linearDNA (genomic) 14 GTGGGATCCC CGGGCAGAGC TGCAGGGC 28 33 base pairs nucleicacid single linear DNA (genomic) 15 GTGGGATCCT TATTACAGCA GTTTCAATGC ACC33 129 base pairs nucleic acid single linear DNA (genomic) 16 GCGGGATCCGCCACCATGAA CTCCTTCTCC ACAAGCGCCT TCGGTCCAGT TGCCTTCTCC 60 CTGGGGCTGCTCCTGGTGTT GCCTGCTGCC TTCCCTGCCC CAGTTGTGAG ACAAGGGGAC 120 CTGGCCAGC 12930 base pairs nucleic acid single linear DNA (genomic) 17 GTGGGATCCTTACAGCAGTT TCAATGCACC 30

What is claimed is:
 1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding the Neutrokine-α polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the Neutrokine-α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22, 1996; (c) a nucleotide sequence encoding the Neutrokine-α polypeptide extracellular domain; (d) a nucleotide sequence encoding the Neutrokine-α polypeptide transmembrane domain; (e) a nucleotide sequence encoding the Neutrokine-α polypeptide intracellular domain; (f) a nucleotide sequence encoding a soluble Neutrokine-α polypeptide comprising the extracellular and intracellular domains but lacking the transmembrane domain; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.
 2. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence in FIG. 1 (SEQ ID NO:1).
 3. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in FIG. 1 (SEQ ID NO:1) encoding the Neutrokine-α polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2).
 4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a soluble Neutrokine-α polypeptide comprising the extracellular domain shown in FIG. 1 (SEQ ID NO:2).
 5. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues n-285 of SEQ ID NO:2, where n is an integer in the range of 2-190 (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues 1-m of SEQ ID NO:2, where m is an integer in the range of 274-284; (c) a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues n-m of SEQ ID NO:2, where n and m are integers as defined respectively in (a) and (b) above; and (d) a nucleotide sequence encoding a polypeptide consisting of a portion of the complete Neutrokine-α amino acid sequence encoded by the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22, 1996 wherein said portion excludes from 1 to 190 amino acids from the amino terminus and from 1 to 11 amino acids from the C-terminus of said complete amino acid sequence.
 6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
 1996. 7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding the Neutrokine-α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
 1996. 8. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a soluble Neutrokine-α polypeptide comprising the extracellular domain encoded by the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
 1996. 9. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e) or (f) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
 10. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Neutrokine-α polypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or (f) of claim
 1. 11. The isolated nucleic acid molecule of claim 10, which encodes an epitope-bearing portion of a Neutrokine-α polypeptide selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe-115 to about Leu-147 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ile-150 to about Tyr-163 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-171 to about Phe-194 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Glu-223 to about Tyr-247 (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about Ser-271 to about Phe-278 (SEQ ID NO:2).
 12. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.
 13. A recombinant vector produced by the method of claim
 12. 14. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 13 into a host cell.
 15. A recombinant host cell produced by the method of claim
 14. 16. A recombinant method for producing a Neutrokine-α polypeptide, comprising culturing the recombinant host cell of claim 15 under conditions such that said polypeptide is expressed and recovering said polypeptide.
 17. An isolated Neutrokine-α polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) the amino acid sequence of the Neutrokine-α polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of the Neutrokine-α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC No. 97768 deposited on Oct. 22, 1996; (c) the amino acid sequence of the Neutrokine-α polypeptide extracellular domain; (d) the amino acid sequence of the Neutrokine-α polypeptide transmembrane domain; (e) the amino acid sequence of the Neutrokine-α polypeptide intracellular domain; (f) the amino acid sequence of a soluble Neutrokine-α polypeptide comprising the domain; and (g) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e) or (f).
 18. An isolated polypeptide of claim 17 comprising an epitope-bearing portion of the Neutrokine-α protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe-115 to about Leu-147 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ile-150 to about Tyr-163 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-171 to about Phe-194 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Glu-223 to about Tyr-247 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-271 to about Phe-278 (SEQ ID NO:2).
 19. An isolated antibody that binds specifically to a Neutrokine-α polypeptide of claim
 17. 20. A pharmaceutical composition comprising a polypeptide of claim 17 and a pharmaceutically acceptable carrier.
 21. An isolated polynucleotide encoding a modified Neutrokine-α protein, wherein, except for at least one conservative amino acid substitution, said modified peptide has an amino acid sequence that is identical to a member selected from the group consisting of: (a) amino acids 1 to 285 of SEQ ID NO:2; (b) amino acids 2 to 285 of SEQ ID NO:2; (c) amino acids 1 to 46 of SEQ ID NO:2; (c) amino acids 47 to 72 of SEQ ID NO:2; and (c) amino acids 73 to 286 of SEQ ID NO:2.
 22. A modified Neutrokine-α polypeptide molecule, wherein, except for at least one conservative amino acid substitution, said modified peptide has an amino acid sequence that is identical to a member selected from the group consisting of: (a) amino acids 1 to 285 of SEQ ID NO:2; (b) amino acids 2 to 285 of SEQ ID NO:2; (c) amino acids 1 to 46 of SEQ ID NO:2; (c) amino acids 47 to 72 of SEQ ID NO:2; and (c) amino acids 73 to 286 of SEQ ID NO:2.
 23. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO:7; (b) the nucleotide sequence of SEQ ID NO:8; (c) the nucleotide sequence of SEQ ID NO:9; (d) the nucleotide sequence of a portion of the sequence shown in FIG. 1 (SEQ ID NO:1) wherein said portion comprises at least 30 contiguous nucleotides from nucleotide 1 to nucleotide 2442, excluding the sequence from nucleotide 1387 to 1421, the sequence from nucleotide 9 to 382, the sequence from nucleotide 1674 to 1996, the sequence from nucleotide 1401 to 1784, the sequence from nucleotide 900 to 1237, and any fragments located within these sequences; and (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above. 